Genetics Lecture 8.PDF

MD210 Genetics Lecture 8 – Clotting disorders and Pharmacogenetics Case Presentation - Ben, second child born to parents Richard and Jenny - Bumped head created a large cephalohematoma - Jenny's two maternal uncles have mobility problems due to haemarthrosis in their knee and ankle joints - Jenny's...

MD210 Genetics Lecture 8 – Clotting disorders and Pharmacogenetics Case Presentation - Ben, second child born to parents Richard and Jenny - Bumped head created a large cephalohematoma - Jenny's two maternal uncles have mobility problems due to haemarthrosis in their knee and ankle joints - Jenny's mother and sister are fine - Ben's sister is also fine X-linked recessive – haemophilia – but which type? Ben's coagulation test results: - Bleeding time = 5 min - Prothrombin time (PT/INR) = 1.0 (measure of extrinsic pathway – measured in a ration of patient to “normal”) - Partial Thromboplastin Time (PTT) = 50 sec (slightly raised) - F VIII activity = 0.5% - F IX activity = 90% - What has he got? Haemophilia A – characterised by F8 deficiency Explainer – coagulation test - Partial thromboplastin time (PTT) (intrinsic/common pathway) o Time it takes plasma to clot when activator (e.g. silica) is added o Normal = 20-35 sec - Prothrombin Time (PT)/international normalised ratio (INR) (extrinsic) o Time it takes plasma to clot after addition of tissue factor o The INR is a standardized measure of this time, which compares patient’s PT to the PT of a normal control sample o Normal = 0.8-1.2 Haemophilia A - Genotype & Phenotype - For each male proband there is a mean of 5 females requesting carrier status determination - Where do we start? - Phenotypic tests may help - measure F8 activity, PTT (intrinsic/common), PT/INR (extrinsic) - “Carrier” females may have disordered coagulation tests or even mild clinical bleeding in tendency (skewed X inactivation) – approx. 30% - Rarely females have severe haemophilia due to skewed X-chromosome inactivation or a disorder of inactivation of X chromosome Haemophilia A & Carrier Women - F8 allele is expressed from whichever X chromosome is active in a given liver cell - One functional F8 allele in most women = 50% activity - Phenotype = clotting - About 10% of carrier females have <40% activity, at risk for abnormal bleeding - 2 non-functional F8 alleles = no functional gene - Phenotype = bleeding Haemophilia A - Assessing Carrier Status: - Genetic methods: 1. Direct sequencing of F8 (a big job but rapidly getting less so) 2. Linkage Markers within F8 o Dinucleotide repeat in intron 13 and 22 (microsatellites) o Known SNP’s in introns 18 and 22 o Need to compare to affected family members Problem A 10 week pregnant female who is a carrier of a Haemophilia A mutation in F8 comes to your clinic to request prenatal testing of her baby. What should you do? - Amniocentesis – risky - Chronic villous sampling – too intrusive - Determine gender of baby – males more likely to be affected Prenatal testing - Initially – test maternal blood for Y Chromosome DNA at 10 weeks - If Y is not detected then chronic villus sampling (1% risk of loss of pregnancy) not required - If Y detected the person may request chronic villus sampling (advantage: prepare for birth of baby – use precautions) - What else is testing for Y Chr used for? Turner Syndrome Case Presentation - James 22 - As a neonate suffered a cephalohaematoma - F IX activity at birth 0.5% (FIX associated with haemophilia B – but doesn’t usually get better) - Increased to 3% at 1 year - 19% at 12 years - No bleeding - 32% at 18 years - Mutational analysis revealed (c.-35G>A) (translation – typically promotor region of gene) - What has he got? Factor IX Leyden - A rare Haemophilia B sub-type (~3%) - Androgen therapy or puberty rises F IX activity from <1% to 30% -60% (fine for a normal clotting phenotype) - Associated mutations (>20) from -40 to + 20 of F IX - This promoter sequence resembles an androgen response element Case Presentation - Carla 23 - Suffers labrum tear during match - Bleeding is profuse during surgery to repair prompting investigation - Coagulation tests: o FVIII – 60% o FIX – 95% o PTT – 40 secs o PT/INR – 1.0 (INR = international normalised ratio) o Bleeding time 20 min - What has she got? Von Willebrand disease (FVIII, associated with adhesion of platelets – bleeding time) Von Willebrand Factor - In plasma, VWF and FVIII circulate as noncovalent complex that regulates platelet aggregation and clot formation - VWF is required for platelet adhesion to subendothelium, and for normal FVIII survival in circulation - VWF protein is not an enzyme - Binds to and inactivates FVIII – increasing serum half-life from 1-2h to 8-12h - Thrombin releases FVIII from VWF and activates platelet receptors so VWF and activates platelet receptors so VWF can bind, facilitating platelet adhesion Von Willebrand Disease - Most frequent congenital clotting disorder (1% prevalence) - VWD Chr 12p13.3 - large gene – 52 exons 178 Kbp - Many alleles - heterogeneity of subtypes - Quantitative or qualitative defects of VWF - Inheritance can be dominant or recessive - Broad clinical phenotype spectrum: - Asymptomatic/mild (common) to severe haemorrhaging (rare) - Treatment – Desmopressin or “factor” - Often reduced FVIII levels: (but not to same extent as Haemophilia A) - Metabolised faster without VWF - Also - faulty thrombocytic adhesion > increased bleeding time Thromboembolism - Formation of thrombus in deep veins (typically of leg) - Pain & Swelling of the leg - Thrombus may become detached from the vein (embolus) and travel to the pulmonary artery - Embolus wedged in pulmonary artery – obstructs blood supply to a segment of lung – pulmonary - Obstructs main pulmonary artery – sudden death (too much clotting instead of not enough) Factor V Leiden - Dutch city of Leiden, late 1980s early 1990s - Investigation of venous thromboembolism in patients without identifiable risk factors - Identified a group of people with poor response to activated protein C (APC) in a coagulation assay Factor V Leiden Thrombophilia - Found a 1601G>A transition in exon 10 of the F5 gene on Ch1q23 - p. Arg506Gln in Factor V (R506Q) - (variant known as FV Leiden) - Pro-coagulant activity of FV is limited by Activated Protein C (APC) which cleaves at the arginine (R) - FV Leiden resistant to APC cleavage (APC could no longer inhibit FV) o Excess conversion of prothrombin to thrombin - What category of mutation is this? Mis-sense - FV Leiden allele frequency ~5% in Caucasians (founder effect?) - Heterozygotes 6 – 7 fold increase in relative risk of venous thromboembolism - Homozygotes up to 80-fold increase in relative risk of thromboembolism - Further increased risk of TE disease with Oral Contraceptives – Why? Oestrogen – increases conc. of different factors Warfarin Anticoagulant - Vitamin K antagonist - Used to manage patients at risk of thromboembolic disease - Too much warfarin can be associated with life threatening hemorrhage - Monitor with coagulation tests (PT/INR) - Adjust dose to maintain within therapeutic window Problem 1 You are treating a patient at risk of thromboembolism. The PT/INR is lower than normal. Would you give warfarin? PT/INR measures time taken for blood to clot in this patient compared to a normal persons. PT/INR is lower than normal = too much clotting factor – clotting faster. So yes, would give warfarin Problem 2 The patient has a CYP2C9*2 allele which inhibits warfarin metabolism. Are they likely to need more or less warfarin than usual? CYP450 system allele – gets rid of drugs from the body – warfarin will accumulate because not being metabolised – more warfarin in blood – higher anticoagulation effect – risk of bleeding Things to remember 1. Factor V Leiden is a relatively common inherited tendency to develop venous thrombosis (Ch1) 2. VWD is a very prevalent, but usually mild, heritable bleeding disorder characterized by prolonged bleeding time. It is associated with heterogeneous quantitative or qualitative defects of VWF and inheritance can be dominant or recessive 3. Pharmacogenetics relates to the idea of targeting drug therapy to maximise benefits and limit adverse effects based on individual genetic profile 4. Optimal dose of warfarin (anticoagulant) is related to genotype (CYP2C9 alleles)